Construction of upp deletion mutant strains of Lactobacillus casei and Lactococcus lactis based on counterselective system using temperature-sensitive plasmid
Construction of upp deletion mutant strains of Lactobacillus casei and Lactococcus lactis based on counterselective system using temperature-sensitive plasmid
复制标题
基于温度敏感质粒反选择系统构建干酪乳杆菌和乳酸乳球菌upp缺失突变株
DOI：
10.1016/j.mimet.2014.04.011
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发表时间：
2014-07-01
影响因子：
2.2
通讯作者：
Li, Yijing
中科院分区：
文献类型：
--
作者：
Song, Li;Cui, Hongyu;Li, Yijing
整合质粒通常用于构建染色体突变，因为它能够通过整合或替换在任何位置改变基因。食品级整合载体可以在不引入任何选择标记或残基的情况下整合到宿主基因组中，重组通常发生在非编码区。本研究利用温度敏感型复制子pWV 01构建了2个氯霉素抗性整合质粒pGBHC 32-upp，该质粒含有尿嘧啶磷酸核糖转移酶（upp）基因作为干酪乳杆菌（Lactobacillus casei，L. casei）ATCC 393和乳酸乳球菌（Lactococcus lactis）MG 1363。然后，我们将设计的同源臂连接到pGBHC 32-upp质粒上，使其整合到细菌染色体上，并选择干酪乳杆菌ATCC 393和L.在5-氟尿嘧啶（5-FU）的存在下，在乳酸MG 1363中进行。遗传稳定性、生长曲线、碳源利用率和扫描电镜分析表明，除5-FU抗性外，野生型和突变型乳酸菌之间没有显著差异。整合系统和upp缺失菌株可用于干酪乳杆菌ATCC 393和乳酸乳杆菌MG 1363染色体上任何位置的基因插入或缺失，并且同源重组不会引入任何选择标记或残留碱基。这些突变株可以进一步研究用于异源蛋白表达和活粘膜疫苗载体的构建。(C)2014爱思唯尔有限公司版权所有。
Integration plasmids are often used in constructing chromosomal mutations, as it enables the alternation of genes at any location by integration or replacement. Food-grade integration vectors can integrate into the host genome without introducing any selectable markers or residual bases, and the recombination often happens in non-coding region. In this study we used the temperature-sensitive pWV01 replicon to construct 2 chloramphenicol-resistant integration plasmids (pGBHC32-upp) containing the uracil phosphoribosyl transferase (upp) gene as a counterselective marker for Lactobacillus casei (L. casei) ATCC393 and Lactococcus lactis (L lactis) MG1363. We then ligated the designed homologous arms to the pGBHC32-upp plasmids to allow their integration to the bacterial chromosome, and selected upp deletion mutants of L casei ATCC393 and L. lactis MG1363 in the presence of 5-fluorouracil (5-FU). Analysis of genetic stability, growth curve, carbon utilization and scanning electronic microscopy showed that, except for 5-FU resistance, there were no significant differences between the wild type and mutant lactic acid bacteria. The integration system and the upp deletion strains could be used in the insertion or deletion of genes at any location of the chromosome of both L casei ATCC 393 and L lactis MG1363, and the homologous recombination would not introduce any selectable markers or residual bases. These mutant strains can be further investigated for heterologous protein expression and construction of a live mucosal vaccine carrier. (C) 2014 Elsevier B.V. All rights reserved.